Targeting pancreatic and ovarian carcinomas using the auristatin-based anti-CD70 antibody-drug conjugate SGN-75

Abstract

Background: CD70 is an ideal target for antibody-based therapies because of its aberrant high expression in renal carcinomas and non-Hodgkin lymphomas and its highly restricted expression in normal tissues. The expression profiling of CD70 in carcinomas has been limited because of the lack of a CD70-specific reagent that works in formalin-fixed paraffin-embedded (FFPE) tissues.

Methods: We generated murine monoclonal antibodies (mAbs) specific for CD70 and validated their specificity by western blot analysis and developed a protocol for immunohistochemistry on FFPE tissues. CD70+ tumour cell lines were used for testing the anti-tumour activity of the anti-CD70 antibody-drug conjugate, SGN-75.

Results: We report novel detection of CD70 expression in multiple cancers including pancreatic (25%), larynx/pharynx (22%), melanoma (16%), ovarian (15%), lung (10%), and colon (9%). Our results show that pancreatic and ovarian tumour cell lines, which express high levels of endogenous or transfected CD70, are sensitive to the anti-tumour activity of SGN-75 in vitro and in vivo.

Conclusion: Development of murine mAbs for robust and extensive screening of FFPE samples coupled with the detection of anti-tumour activity in novel indications provide rationale for expanding the application of SGN-75 for the treatment of multiple CD70 expressing cancers.

Figure 1

Characterisation of anti-CD70 1C1 and 5D12. (A) Western blots containing FLAG-tagged CD70 ECD (lanes 1 and 5) and membrane extracts from HEK 293 cells (lanes 2 and 6), HEK 293:CD70 transfectants (lanes 3 and 7) and 786-O cells (lanes 4 and 8) were reacted with mAb 1C1 (left panel) or mAb 5D12 (right panel). The mAb 1C1 and 5D12 specifically bound recombinant CD70 (lane 1 and 4) and three distinct bands (^*) from CD70+ membrane extracts (lanes 3, 4, 7, and 8). Extracts from parental HEK 293 cells were negative as expected (lane 2 and 6). The β-actin control shows comparable quantities of protein were loaded for each membrane extract (lower panels). (B) IHC analysis on FFPE normal human tissues showing limited expression of CD70 in lymphoid tissues. Red stain indicates presence of CD70+ cells. 1C1 mAb was used for these set of images. × 400 magnification, scale bar=50 μm.

Figure 2

Correlation of CD70 expression in cell lines by qFACS, IHC, and reverse trancriptase–PCR: (A) Quantitative flow cytometric analyses were carried out on ovarian (SK-OV-3, TOV-21G), and pancreatic (Panc-1) cell lines using a PE-conjugate murine anti-CD70 ab (BD Pharmingen). CD70 copy number was determined using standard labelled bead controls (QiFiKit, DAKO). (B) Corresponding cell pellets were harvested and processed as FFPE samples and immunohistochemistry using 1C1 (as shown) or 5D12 was carried out using alkaline phosphatase-labelled secondary antibody and Fast Red as the chromogen. Images were taken using a Zeiss microscope at 400 × magnification. Scale bar=50 μm. (C) Detection of human CD70 expression by reverse trancriptase–PCR: expression of CD70 in various CD70+ cell lines was determined using cDNA preparations obtained from the cells (lanes 1: AN3CA ovarian; 2: SKOV-3 ovarian; 3: TOV-21G ovarian; 4: NCI-H716 colorectal; 5: 786-O renal; 6: PANC-1 pancreatic; 7: IGROV-1 ovarian (CD70 negative); 8: H₂O). Primers that partially spanned the coding regions were used for CD70. PCR products were sequenced to confirm their identity. hCD70 (TNFSF7) fragment=852 bp, including exons 1,2, and 3. Human GAPDH (hGAPDH) (580 bp) was used as housekeeping gene PCR control.

Figure 3

Immunohistochemistry analysis using anti-CD70 mAb (1C1) in tumour microarrays. CD70 expression in pancreatic (A) and ovarian (B) tumours is shown. Of the 140 and 241 individual cases of pancreatic and ovarian carcinoma, respectively, 35 cases (25%) and 37 cases (15%) were found to be CD70+. In this analysis, the area of transformed cells within each biopsy stained positively for CD70 expression is categorised into <25%, 25–50%, 50–75%, and >75%. The intensity of CD70 expression is expressed as: 1=weak, 2=mild, 3=moderate, 4=strong ( × 400 magnification). To illustrate the heterogeneity in CD70 expression, the percent area positive is plotted against staining intensity. In these plots, the number in each in the graphs denotes the number of CD70+ cases. Representative images for low, heterogeneous (25–50% positive, 2+) and high, homogeneous (>75% positive, 4+) CD70 for pancreatic and ovarian carcinoma are shown.

Figure 4

In vitro and In vivo Anti-tumour Activity of SGN-75: (A and B) In vitro cytotoxicity assay showing potency of SGN-75 (triangles) on SK-OV-3 ovarian and CD70-transfected MiaPaCa-2 pancreatic carcinoma cell lines. The controls, unconjugated h1F6 control () and non-binding ADC (A, squares) have no effect. IC₅₀ for SGN-75 and non-binding ADC are noted. Similarly, no cytotoxicity is observed in the untransfected MiaPaCa-2 control (B, squares). (C and D) In vivo anti-tumour activity of SGN-75 in0-transfected MiaPaCa-2 pancreatic carcinoma tumours in nude mice. Mice treated with SGN-75 (squares) showed a significantly reduced median tumour volume (C) and a higher percent survival (D) when compared with untreated (circles) and non-binding ADC (triangles).